RESEARCH ARTICLE
Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells Maria Kawalec1☯, Anna Boratyńska-Jasińska1☯, Małgorzata Beręsewicz1, Dorota Dymkowska2, Krzysztof Zabłocki2‡*, Barbara Zabłocka1‡ 1 Molecular Biology Unit, Mossakowski Medical Research Centre, PAS, Warsaw, Poland, 2 Department of Biochemistry, Nencki Institute of Experimental Biology, PAS, Warsaw, Poland ☯ These authors contributed equally to this work. ‡ Senior co-authors. * [email protected]
Abstract OPEN ACCESS Citation: Kawalec M, Boratyńska-Jasińska A, Beręsewicz M, Dymkowska D, Zabłocki K, Zabłocka B (2015) Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells. PLoS ONE 10(7): e0134162. doi:10.1371/ journal.pone.0134162 Editor: Francesco Cappello, University of Palermo, ITALY Received: April 4, 2015 Accepted: July 6, 2015 Published: July 31, 2015 Copyright: © 2015 Kawalec et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by Narodowe Centrum Nauki grant number NN402474640. BioTech Poland Sp. z o.o. provided the access to the xCELLigence RTCA DP Instrument. Competing Interests: BioTech Poland Sp. z o.o. provided the access to the xCELLigence RTCA DP instrument. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.
Introduction Mitofusin (Mfn2) is encoded by nuclear DNA. It locates mainly in the outer mitochondrial membrane, and is involved in its rearrangement [1]. Moreover, Mfn2 is also present in the
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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Mitofusin, Mitochondria and Energy Metabolism in MEF Cells
endoplasmic reticulum membrane and plays a role in ER interactions with mitochondria [2]. Growing interest of Mfn2 as a protein putatively involved in cell survival, begun when changes in Mfn2 expression in muscles of diabetic patients [3] and in blood vessels of patients suffering from vascular proliferative disorders [4] were observed. Moreover, a significant point in studies of mitofusin 2 is to understand the link between MFN2 mutations and Charcot-Marie-Tooth type 2A axonal neuropathy development [5]. Mitofusin 2 is engaged in the proper formation of mitochondrial network. Fusion of mitochondria might be regarded as a way to equal distribution of mtDNA in the cells and, as a consequence, proper formation of mitochondrial complexes [6]. There are conflicting data on the composition and activity of respiratory complexes and ATP level in various tissues from CMT patients and experimental models in vitro. Vielhaber et al. (2012) showed a slight decrease in the activity of the respiratory complex I in fibroblasts and in the muscle fibers biopsies from CMT2A patients [7]. Earlier it was shown that mitochondrial respiratory enzyme activity was normal in skeletal muscle mitochondria from a CMT2 patient with an MFN2 mutation and ATP production was also unaffected [8]. Finding the molecular mechanism of neuronal mitochondria metabolism and axonal loss observed in CMT2A in the absence of fully functional MFN2 would be crucial for CMT2A counseling and also desirable for therapeutic strategies of wide range of disorders. Therefore, investigations of cellular energy metabolism in mitofusin 2-depleted cells should be continued to clarify various aspects of its improper functions. Here, energy metabolism and proliferation rate of mitofusin 2-depleted MEF cells and their mitofusin 2-positive equivalents were in focus. Relatively increased rate of oxygen consumption together with increased level of mitochondrial markers, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) proteins and mitochondrial mass but unaffected mtDNA content suggest an increased mitochondrial biogenesis which may efficiently counteract but not outbalance detrimental effects of mitofusin 2 deficiency.
Materials and Methods Cell culture Mouse Embryonic Fibroblasts: Wild type MEFs (MEFwt) (ATCC-CRL-2991), Mfn2-null MEF (MEFMfn2-/-) (ATCC-CRL-2993) were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with either 10% Fetal Bovine Serum (Gibco) or 10% Bovine Calf Serum (Gibco) and antibiotics (100 units/ml penicillin plus streptomycin 100 μg/ ml), in 5% CO2 atmosphere at 37°C.
Cell proliferation assay Cell doubling time was analyzed using xCELLigence RTCA DP instrument (ACEA Biosystems), which was placed in a humidified incubator at 37°C with 5% CO2. The electronic sensors provided a continuous and quantitative measurement of cell index in each well. Cell index represents a quantitative measure of cell number present in a well e.g. low cell index reflects fewer cells attached to the electrodes. Approximately 10 x 103 of MEFs were plated into E-plate 16 and doubling time of cells were analysed based on real time measurements carried out for 48 hours [9,10].
Cellular respiration Cellular respiration was measured polarographically, with the use of Oxygraph-2k (OROBOROS; INSTRUMENTS GmbH, Austria). Cells from 10 cm diameter tissue culture dish were
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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trypsinized and resuspended in pre-warmed PBS to final protein concentration 0.15–0.3 mg/ ml. Oxygen consumption was measured at 37°C in substrate free solution (PBS) and after administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and CCCP (1 μM). After respiration assay, cell suspension was mixed with 0.5 M NaOH (1:1) and protein concentration was measured. Data were presented as mean use of O2 picomoles per milligram of protein per second.
Mitochondrial membrane potential Mitochondrial potential was measured fluorimetrically according to the Cossarizza et al. (2001) [11]. Cells were trypsinized, incubated with 5 μM JC-1 dye (5,5’,6,6-tetrachloro1,1’,3,3-tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes, Invitrogen) at 37°C in the dark for 15 min. Parallel to JC-1 cell also were labeled with the nuclear stain TO-PRO-3 (0.4 μM). When indicated, prior to JC-1/ TO-PRO-3 staining, cells were preincubated for 10 min with 3 μM valinomycin and 5 μM CCCP or 0.1 μg/ml oligomycin. Fluorescence was measured by BD FACSCalibur flow cytometry equipped with argon laser (excitation 488 nm for JC-1) and excitation/emission (nm) 642⁄661 for TO-PRO-3. JC-1 fluorescence was measured only in TO-PRO-3 negative cells.
Blue Native sample preparation, electrophoresis and in-gel complex V activity assay MEFwt and MEFMfn2-/- cells were trypsinized, washed with cold PBS and homogenized in homogenization buffer (10 mM Tris-HCl, 0.25 M sucrose, 2 mM EDTA, 1 mM PMSF, Protease Inhibitor Cocktail from Sigma; pH 7.4) on ice. Homogenates were centrifuged at 1000 x g for 5 min. at 4°C. Supernatants were spun at 10 000 x g for 20 min at 4°C, to obtain pellet enriched in mitochondria. Samples containing 100 μg protein were pelleted and resuspended in 10 μl aminocaproic acid buffer (1.5 M ACA, 50 mM Bis-Tris; pH 7.0) with PMSF and protease inhibitor cocktail (Sigma). 1 μl of 10% dodecylomaltoside was added to each sample. After 30 min incubation on ice, samples were spun at 17 000 x g for 20 min at 4°C. Supernatants were mixed with 0.4 μl aminocaproic acid buffer with 5% Coomassie G-250 (BioRad) and loaded onto 5–12% gradient acrylamide gel. Electrophoresis was started with 70 V in cathode buffer (50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7.0 at 4°C with 0.02% Coomassie G-250) and anode buffer (50 mM Bis-Tris/HCl, pH 7.0 at 4°C). After 1 h, voltage was increased to 200 V. To visualize complex V activity, gel stripes were incubated over night at room temperature in solution containing 35 mM Tris HCl, 270 mM glycine (pH 8.3–8.6), 14 mM MgCl2, 0.1% Pb (NO3)2, 8 mM ATP according to Sabar et al., 2005 and Yan et al., 2009 [12,13]. An intensity of bands corresponds to ATP-ase enzymatic activity.
Western blot analysis (WB) of selected respiratory chain proteins and mitochondrial ATP synthase subunit were performed with MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (MitoScience). Cell extracts were prepared with Lysis Buffer (Cell Signalling), separated by 12% SDS-polyacrylamide gel electrophoresis (30 μg of protein per line) and electro-transferred to nitrocellulose membrane. In WB, following subunits were detected: NDUFB8 (complex I), CII-30 kDa (FeS; complex II); CIII-Core 2 protein (complex III); C-IV-I subunit (complex IV); C-V-alpha subunit (complex V). Data were expressed as relative immunoreactivity of each complex in knockout cells in relation to wild type one. To study the mitochondrial biogenesis the following primary antibodies were used: TFAM (GenWay Biotech, GWB-22C6C2), TOM20 (Santa Cruz Biotechnology, sc-11415), PGC-1α and PCNA, β-actin and GAPDH as loading markers (Santa
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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Cruz Biotechnology, sc-13067, sc-9857, Sigma-Aldrich, A2066 and EMD Millipore, MAB374, respectively).
Mitochondrial mass with NAO Cells were trypsynized and suspended in the fresh culture medium supplemented with 1 μM NAO, incubated in 37°C in the atmosphere of 5% CO2/95% air for 30 minutes, according to [14]. After washing the cells were suspended in PBS with calcium and magnesium ions. Measurements were done in FACS CAlibur with excitation/emission 495/522 nm. In each sample 10 000 events were counted. The data are expressed as a geometric mean of fluorescence ± SD from 6 independent experiments.
ATP content ATP was measured enzymatically as described earlier [15], according to the method of Williamson and Corkey (1969) [16]. Cells were incubated in Krebs—Henseleit solution (10 mM HEPES, 2 mM NaHCO3, 135 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 1 mM pyruvate, pH 7.4) without or with glucose (5.6 mM), ATP synthase inhibitor oligomycin (0.1 μg/ml) or glycolysis inhibitor iodoacetate (1 mM) for 10 min at 37°C. Then the incubation solution was removed and cells were extracted with 3.5% HClO4 for 10 min on ice. Extracts were collected and neutralized with 2 M K2CO3. ATP concentration was measured fluorimetrically, with the use of glucose-6-phosphate dehydrogenase and hexokinase in pH 7.4 buffer (50 mM triethanolamine-HCl, 10 mM MgCl2, 5 mM EDTA). ATP concentration was standardized with the use of ATP solution of known concentration. Protein concentration was measured in cells solubilized in 0.5 M NaOH. Data were presented as ATP concentration (nanomoles per milligram of protein) ± S.D. for 3–5 independent experiments.
Lactate synthesis Lactate synthesis was estimated on a basis of enzymatic measurement of lactate concentration in the acidified incubation medium, according to [17]. It allowed determining both intracellular lactate content and lactate released from cells. The cells were incubated in Krebs—Henseleit solution (10 mM HEPES, 2 mM NaHCO3, 135 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, supplemented with 1 mM pyruvate and 5.6 mM glucose, pH 7.4) for 30 min. Next, the incubation was acidified with HClO4 (final concentration 5%). After 10 minutes on ice extracts were neutralized with 2 M K2CO3 and lactate was measured fluorimetrically, with the use of lactate dehydrogenase in a buffer composed of 0.5 M glycine, 0.4 M hydrazine sulphate, 5 mM EDTA pH 9.5. Lactate concentration was calculated with the use of standardized lactate solution. Protein concentration was measured in cells solubilized in 0.5 M NaOH. Data were presented as lactate content (nanomoles per milligram of protein) ± S. D. for 6 independent experiments.
mtDNA content mtDNA content was measured by real-time PCR, according to [18]. Quantification was based on mtDNA to nuclear thymidylate kinase gene ratio [19]. Total DNA was extracted by Genomic Mini Kit (A&A Biotechnology). PCR amplifications were carried out with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7500 real-time PCR thermal cycler in conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Following primers were used: Mouse mitochondrial displacement loop (Dloop) region (D-loop F, 5’-CCAAAAAACACTAAGAACTTGAAAGACA-3’ and D-loop R,
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5’-GTCA TATTTTGGGAACTACTAGAATTGATC-3’) and single-copy nuclear thymidylate kinase gene (F, 5’-GACTGTATTGAGCGGCTTCAGA-3’ and R, 5’-CATGCTCGGTGTGAG CCATA-3’). Relative mtDNA quantity was determined using ΔΔCt method, according to Livak and Schmittgen (2001) [19].
Protein concentration Protein was measured with the use of Modified Lowry Protein Assay Kit (Pierce).
Statistics Data are shown as mean value ± standard deviation. The statistical significant differences were calculated using Bonferroni or Student’s t-est for the simultaneous analysis of multiple test groups preceded by analysis of variance ANOVA.
Results Cell proliferation Mitofusin-deficient cells were previously found to proliferate faster that their mitofusin 2-positive equivalents and this was attributed to the mitofusin 2-dependent inhibition of the Ras-RafERK signaling pathway [20]. However, in view of inconsistency concerning type of serum for supplementation of the growth media (FBS is commonly used by researchers and BCS is recommended by supplier) which may imply different composition of growth factors, the rate of both cell lines proliferation in the presence of either FBS or BCS in the medium was here determined. It was found that MEFMfn2-/- cells proliferated much faster than MEFwt cells when grown in the FBS supplemented medium while in BCS it was completely reversed; proliferation of the mitofusin 2-deficient cells was substantially slowed-down in comparison to proliferation of the mitofusin–positive fibroblasts. Interestingly, proliferation of the latter was less sensitive to the type of serum (Fig 1).
Fig 1. Doubling time of MEFwt and MEFMfn2-/- cells. The cells were grown in media supplemented with either FBS or BCS. Doubling time of cells were analysed based on real time measurements carried out for 48 hours using xCELLigence RTCA DP instrument. Data show mean values ± S.D. n = 10 * p < 0.05, ** p < 0.01. doi:10.1371/journal.pone.0134162.g001
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Oxygen consumption and mitochondrial membrane potential Rate of oxygen consumption is a complex measure of cellular energy metabolism. It depends on a variety of parameters such as substrate and oxygen availability, cellular energy charge, activity of respiratory complexes and mitochondrial membrane potential. A comparison of mitochondrial respiration in various cells under different experimental conditions allows concluding about differences in their metabolic capability. This is a first test typically performed to characterize bioenergetics state of cells. Here two lines of mouse embryonic fibroblasts MEF have been investigated. They were MEFMfn2-/- and their wild type equivalent (MEFwt) as a control. As shown in Fig 2, MEF cells with deleted Mfn2 gene exhibit substantially faster oxygen consumption than control fibroblasts. This statistically significant difference is very similar in cells incubated without any exogenous substrates as well as in the presence of glucose and pyruvate. It indicates that availability of endogenously stored respiratory substrates does not limit respiration rate in both cell lines tested, thus it cannot be responsible for observed differences. Moreover, the same tendency was found in cells treated with mitochondrial uncoupler. The latter may suggests an increased cellular respiratory capacity presumably due to elevated content of mitochondrial respiratory enzymes, enhanced biogenesis of these organelles or increased enzymatic capacity of respiratory complexes. Indeed, respiratory capacity expressed as a ratio between rate of oxygen consumption in the presence of CCCP to that in the presence oligomycin prior to mitochondrial uncoupling was very similar in both MEFwt and MEFMfn2-/- cells (Table 1) suggesting unaffected specific enzymatic activity of the respiratory chain. More intense oxygen consumption by cells depleted of mitofusin 2 and unaffected respiratory capacity were observed irrespectively of a type of serum supplementing the growth media (Table 1). To exclude cell-line specific effects, the experiments were repeated in a second set of MEFwt and MEFMfn2-/- cells purchased from the same supplier but differing in their batch number, and the same responses were found. Thus a medium-dependent changes of MEFMfn2-/- proliferation seems to be unmatched with faster respiration of mitofusin-deficient cells. High consistency of obtained results excluded randomness of our observations. In further
Fig 2. Oxygen consumption by MEFwt and MEFMfn2-/- cells. The cells were grown in media supplemented with either FBS or BCS. Oxygen consumption was measured polarographically at 37°C in a substrate free buffered saline solution (PBS with Mg2+ and Ca2+), and after sequential administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and 1 μM CCCP. Data are expressed as pmoles O2 x s-1/mg protein and show mean values ± S.D. n = 4; *p
Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells Maria Kawalec1☯, Anna Boratyńska-Jasińska1☯, Małgorzata Beręsewicz1, Dorota Dymkowska2, Krzysztof Zabłocki2‡*, Barbara Zabłocka1‡ 1 Molecular Biology Unit, Mossakowski Medical Research Centre, PAS, Warsaw, Poland, 2 Department of Biochemistry, Nencki Institute of Experimental Biology, PAS, Warsaw, Poland ☯ These authors contributed equally to this work. ‡ Senior co-authors. * [email protected]
Abstract OPEN ACCESS Citation: Kawalec M, Boratyńska-Jasińska A, Beręsewicz M, Dymkowska D, Zabłocki K, Zabłocka B (2015) Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells. PLoS ONE 10(7): e0134162. doi:10.1371/ journal.pone.0134162 Editor: Francesco Cappello, University of Palermo, ITALY Received: April 4, 2015 Accepted: July 6, 2015 Published: July 31, 2015 Copyright: © 2015 Kawalec et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by Narodowe Centrum Nauki grant number NN402474640. BioTech Poland Sp. z o.o. provided the access to the xCELLigence RTCA DP Instrument. Competing Interests: BioTech Poland Sp. z o.o. provided the access to the xCELLigence RTCA DP instrument. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.
Introduction Mitofusin (Mfn2) is encoded by nuclear DNA. It locates mainly in the outer mitochondrial membrane, and is involved in its rearrangement [1]. Moreover, Mfn2 is also present in the
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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Mitofusin, Mitochondria and Energy Metabolism in MEF Cells
endoplasmic reticulum membrane and plays a role in ER interactions with mitochondria [2]. Growing interest of Mfn2 as a protein putatively involved in cell survival, begun when changes in Mfn2 expression in muscles of diabetic patients [3] and in blood vessels of patients suffering from vascular proliferative disorders [4] were observed. Moreover, a significant point in studies of mitofusin 2 is to understand the link between MFN2 mutations and Charcot-Marie-Tooth type 2A axonal neuropathy development [5]. Mitofusin 2 is engaged in the proper formation of mitochondrial network. Fusion of mitochondria might be regarded as a way to equal distribution of mtDNA in the cells and, as a consequence, proper formation of mitochondrial complexes [6]. There are conflicting data on the composition and activity of respiratory complexes and ATP level in various tissues from CMT patients and experimental models in vitro. Vielhaber et al. (2012) showed a slight decrease in the activity of the respiratory complex I in fibroblasts and in the muscle fibers biopsies from CMT2A patients [7]. Earlier it was shown that mitochondrial respiratory enzyme activity was normal in skeletal muscle mitochondria from a CMT2 patient with an MFN2 mutation and ATP production was also unaffected [8]. Finding the molecular mechanism of neuronal mitochondria metabolism and axonal loss observed in CMT2A in the absence of fully functional MFN2 would be crucial for CMT2A counseling and also desirable for therapeutic strategies of wide range of disorders. Therefore, investigations of cellular energy metabolism in mitofusin 2-depleted cells should be continued to clarify various aspects of its improper functions. Here, energy metabolism and proliferation rate of mitofusin 2-depleted MEF cells and their mitofusin 2-positive equivalents were in focus. Relatively increased rate of oxygen consumption together with increased level of mitochondrial markers, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) proteins and mitochondrial mass but unaffected mtDNA content suggest an increased mitochondrial biogenesis which may efficiently counteract but not outbalance detrimental effects of mitofusin 2 deficiency.
Materials and Methods Cell culture Mouse Embryonic Fibroblasts: Wild type MEFs (MEFwt) (ATCC-CRL-2991), Mfn2-null MEF (MEFMfn2-/-) (ATCC-CRL-2993) were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with either 10% Fetal Bovine Serum (Gibco) or 10% Bovine Calf Serum (Gibco) and antibiotics (100 units/ml penicillin plus streptomycin 100 μg/ ml), in 5% CO2 atmosphere at 37°C.
Cell proliferation assay Cell doubling time was analyzed using xCELLigence RTCA DP instrument (ACEA Biosystems), which was placed in a humidified incubator at 37°C with 5% CO2. The electronic sensors provided a continuous and quantitative measurement of cell index in each well. Cell index represents a quantitative measure of cell number present in a well e.g. low cell index reflects fewer cells attached to the electrodes. Approximately 10 x 103 of MEFs were plated into E-plate 16 and doubling time of cells were analysed based on real time measurements carried out for 48 hours [9,10].
Cellular respiration Cellular respiration was measured polarographically, with the use of Oxygraph-2k (OROBOROS; INSTRUMENTS GmbH, Austria). Cells from 10 cm diameter tissue culture dish were
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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trypsinized and resuspended in pre-warmed PBS to final protein concentration 0.15–0.3 mg/ ml. Oxygen consumption was measured at 37°C in substrate free solution (PBS) and after administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and CCCP (1 μM). After respiration assay, cell suspension was mixed with 0.5 M NaOH (1:1) and protein concentration was measured. Data were presented as mean use of O2 picomoles per milligram of protein per second.
Mitochondrial membrane potential Mitochondrial potential was measured fluorimetrically according to the Cossarizza et al. (2001) [11]. Cells were trypsinized, incubated with 5 μM JC-1 dye (5,5’,6,6-tetrachloro1,1’,3,3-tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes, Invitrogen) at 37°C in the dark for 15 min. Parallel to JC-1 cell also were labeled with the nuclear stain TO-PRO-3 (0.4 μM). When indicated, prior to JC-1/ TO-PRO-3 staining, cells were preincubated for 10 min with 3 μM valinomycin and 5 μM CCCP or 0.1 μg/ml oligomycin. Fluorescence was measured by BD FACSCalibur flow cytometry equipped with argon laser (excitation 488 nm for JC-1) and excitation/emission (nm) 642⁄661 for TO-PRO-3. JC-1 fluorescence was measured only in TO-PRO-3 negative cells.
Blue Native sample preparation, electrophoresis and in-gel complex V activity assay MEFwt and MEFMfn2-/- cells were trypsinized, washed with cold PBS and homogenized in homogenization buffer (10 mM Tris-HCl, 0.25 M sucrose, 2 mM EDTA, 1 mM PMSF, Protease Inhibitor Cocktail from Sigma; pH 7.4) on ice. Homogenates were centrifuged at 1000 x g for 5 min. at 4°C. Supernatants were spun at 10 000 x g for 20 min at 4°C, to obtain pellet enriched in mitochondria. Samples containing 100 μg protein were pelleted and resuspended in 10 μl aminocaproic acid buffer (1.5 M ACA, 50 mM Bis-Tris; pH 7.0) with PMSF and protease inhibitor cocktail (Sigma). 1 μl of 10% dodecylomaltoside was added to each sample. After 30 min incubation on ice, samples were spun at 17 000 x g for 20 min at 4°C. Supernatants were mixed with 0.4 μl aminocaproic acid buffer with 5% Coomassie G-250 (BioRad) and loaded onto 5–12% gradient acrylamide gel. Electrophoresis was started with 70 V in cathode buffer (50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7.0 at 4°C with 0.02% Coomassie G-250) and anode buffer (50 mM Bis-Tris/HCl, pH 7.0 at 4°C). After 1 h, voltage was increased to 200 V. To visualize complex V activity, gel stripes were incubated over night at room temperature in solution containing 35 mM Tris HCl, 270 mM glycine (pH 8.3–8.6), 14 mM MgCl2, 0.1% Pb (NO3)2, 8 mM ATP according to Sabar et al., 2005 and Yan et al., 2009 [12,13]. An intensity of bands corresponds to ATP-ase enzymatic activity.
Western blot analysis (WB) of selected respiratory chain proteins and mitochondrial ATP synthase subunit were performed with MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (MitoScience). Cell extracts were prepared with Lysis Buffer (Cell Signalling), separated by 12% SDS-polyacrylamide gel electrophoresis (30 μg of protein per line) and electro-transferred to nitrocellulose membrane. In WB, following subunits were detected: NDUFB8 (complex I), CII-30 kDa (FeS; complex II); CIII-Core 2 protein (complex III); C-IV-I subunit (complex IV); C-V-alpha subunit (complex V). Data were expressed as relative immunoreactivity of each complex in knockout cells in relation to wild type one. To study the mitochondrial biogenesis the following primary antibodies were used: TFAM (GenWay Biotech, GWB-22C6C2), TOM20 (Santa Cruz Biotechnology, sc-11415), PGC-1α and PCNA, β-actin and GAPDH as loading markers (Santa
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Cruz Biotechnology, sc-13067, sc-9857, Sigma-Aldrich, A2066 and EMD Millipore, MAB374, respectively).
Mitochondrial mass with NAO Cells were trypsynized and suspended in the fresh culture medium supplemented with 1 μM NAO, incubated in 37°C in the atmosphere of 5% CO2/95% air for 30 minutes, according to [14]. After washing the cells were suspended in PBS with calcium and magnesium ions. Measurements were done in FACS CAlibur with excitation/emission 495/522 nm. In each sample 10 000 events were counted. The data are expressed as a geometric mean of fluorescence ± SD from 6 independent experiments.
ATP content ATP was measured enzymatically as described earlier [15], according to the method of Williamson and Corkey (1969) [16]. Cells were incubated in Krebs—Henseleit solution (10 mM HEPES, 2 mM NaHCO3, 135 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 1 mM pyruvate, pH 7.4) without or with glucose (5.6 mM), ATP synthase inhibitor oligomycin (0.1 μg/ml) or glycolysis inhibitor iodoacetate (1 mM) for 10 min at 37°C. Then the incubation solution was removed and cells were extracted with 3.5% HClO4 for 10 min on ice. Extracts were collected and neutralized with 2 M K2CO3. ATP concentration was measured fluorimetrically, with the use of glucose-6-phosphate dehydrogenase and hexokinase in pH 7.4 buffer (50 mM triethanolamine-HCl, 10 mM MgCl2, 5 mM EDTA). ATP concentration was standardized with the use of ATP solution of known concentration. Protein concentration was measured in cells solubilized in 0.5 M NaOH. Data were presented as ATP concentration (nanomoles per milligram of protein) ± S.D. for 3–5 independent experiments.
Lactate synthesis Lactate synthesis was estimated on a basis of enzymatic measurement of lactate concentration in the acidified incubation medium, according to [17]. It allowed determining both intracellular lactate content and lactate released from cells. The cells were incubated in Krebs—Henseleit solution (10 mM HEPES, 2 mM NaHCO3, 135 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, supplemented with 1 mM pyruvate and 5.6 mM glucose, pH 7.4) for 30 min. Next, the incubation was acidified with HClO4 (final concentration 5%). After 10 minutes on ice extracts were neutralized with 2 M K2CO3 and lactate was measured fluorimetrically, with the use of lactate dehydrogenase in a buffer composed of 0.5 M glycine, 0.4 M hydrazine sulphate, 5 mM EDTA pH 9.5. Lactate concentration was calculated with the use of standardized lactate solution. Protein concentration was measured in cells solubilized in 0.5 M NaOH. Data were presented as lactate content (nanomoles per milligram of protein) ± S. D. for 6 independent experiments.
mtDNA content mtDNA content was measured by real-time PCR, according to [18]. Quantification was based on mtDNA to nuclear thymidylate kinase gene ratio [19]. Total DNA was extracted by Genomic Mini Kit (A&A Biotechnology). PCR amplifications were carried out with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7500 real-time PCR thermal cycler in conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Following primers were used: Mouse mitochondrial displacement loop (Dloop) region (D-loop F, 5’-CCAAAAAACACTAAGAACTTGAAAGACA-3’ and D-loop R,
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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Mitofusin, Mitochondria and Energy Metabolism in MEF Cells
5’-GTCA TATTTTGGGAACTACTAGAATTGATC-3’) and single-copy nuclear thymidylate kinase gene (F, 5’-GACTGTATTGAGCGGCTTCAGA-3’ and R, 5’-CATGCTCGGTGTGAG CCATA-3’). Relative mtDNA quantity was determined using ΔΔCt method, according to Livak and Schmittgen (2001) [19].
Protein concentration Protein was measured with the use of Modified Lowry Protein Assay Kit (Pierce).
Statistics Data are shown as mean value ± standard deviation. The statistical significant differences were calculated using Bonferroni or Student’s t-est for the simultaneous analysis of multiple test groups preceded by analysis of variance ANOVA.
Results Cell proliferation Mitofusin-deficient cells were previously found to proliferate faster that their mitofusin 2-positive equivalents and this was attributed to the mitofusin 2-dependent inhibition of the Ras-RafERK signaling pathway [20]. However, in view of inconsistency concerning type of serum for supplementation of the growth media (FBS is commonly used by researchers and BCS is recommended by supplier) which may imply different composition of growth factors, the rate of both cell lines proliferation in the presence of either FBS or BCS in the medium was here determined. It was found that MEFMfn2-/- cells proliferated much faster than MEFwt cells when grown in the FBS supplemented medium while in BCS it was completely reversed; proliferation of the mitofusin 2-deficient cells was substantially slowed-down in comparison to proliferation of the mitofusin–positive fibroblasts. Interestingly, proliferation of the latter was less sensitive to the type of serum (Fig 1).
Fig 1. Doubling time of MEFwt and MEFMfn2-/- cells. The cells were grown in media supplemented with either FBS or BCS. Doubling time of cells were analysed based on real time measurements carried out for 48 hours using xCELLigence RTCA DP instrument. Data show mean values ± S.D. n = 10 * p < 0.05, ** p < 0.01. doi:10.1371/journal.pone.0134162.g001
PLOS ONE | DOI:10.1371/journal.pone.0134162 July 31, 2015
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Mitofusin, Mitochondria and Energy Metabolism in MEF Cells
Oxygen consumption and mitochondrial membrane potential Rate of oxygen consumption is a complex measure of cellular energy metabolism. It depends on a variety of parameters such as substrate and oxygen availability, cellular energy charge, activity of respiratory complexes and mitochondrial membrane potential. A comparison of mitochondrial respiration in various cells under different experimental conditions allows concluding about differences in their metabolic capability. This is a first test typically performed to characterize bioenergetics state of cells. Here two lines of mouse embryonic fibroblasts MEF have been investigated. They were MEFMfn2-/- and their wild type equivalent (MEFwt) as a control. As shown in Fig 2, MEF cells with deleted Mfn2 gene exhibit substantially faster oxygen consumption than control fibroblasts. This statistically significant difference is very similar in cells incubated without any exogenous substrates as well as in the presence of glucose and pyruvate. It indicates that availability of endogenously stored respiratory substrates does not limit respiration rate in both cell lines tested, thus it cannot be responsible for observed differences. Moreover, the same tendency was found in cells treated with mitochondrial uncoupler. The latter may suggests an increased cellular respiratory capacity presumably due to elevated content of mitochondrial respiratory enzymes, enhanced biogenesis of these organelles or increased enzymatic capacity of respiratory complexes. Indeed, respiratory capacity expressed as a ratio between rate of oxygen consumption in the presence of CCCP to that in the presence oligomycin prior to mitochondrial uncoupling was very similar in both MEFwt and MEFMfn2-/- cells (Table 1) suggesting unaffected specific enzymatic activity of the respiratory chain. More intense oxygen consumption by cells depleted of mitofusin 2 and unaffected respiratory capacity were observed irrespectively of a type of serum supplementing the growth media (Table 1). To exclude cell-line specific effects, the experiments were repeated in a second set of MEFwt and MEFMfn2-/- cells purchased from the same supplier but differing in their batch number, and the same responses were found. Thus a medium-dependent changes of MEFMfn2-/- proliferation seems to be unmatched with faster respiration of mitofusin-deficient cells. High consistency of obtained results excluded randomness of our observations. In further
Fig 2. Oxygen consumption by MEFwt and MEFMfn2-/- cells. The cells were grown in media supplemented with either FBS or BCS. Oxygen consumption was measured polarographically at 37°C in a substrate free buffered saline solution (PBS with Mg2+ and Ca2+), and after sequential administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and 1 μM CCCP. Data are expressed as pmoles O2 x s-1/mg protein and show mean values ± S.D. n = 4; *p
